During development, cells progressively lose the ability to differentiate into multiple cell types. During the first stage of development cells are totipotent and able to give rise to all cells of an organism. As development continues, cells differentiate and become pluripotent (able to differentiate into all cells of the embryo except trophoblast). As the embryo develops, cells become multipotent (able to give rise to a particular cell type of a given lineage) or unipotent (terminally differentiated and only able to give rise to a single cell type or lineage). Pluripotent cells can maintain prolonged, undifferentiated proliferation (self-renewal). In humans, they have the potential to form any one of the 220 different cell types.
Upon fertilization, each cell in an organism carries identical genetic material known as genomic DNA. As an organism develops, the expression of genetic material is altered by internal and external environmental factors that decide their fate in the organism. The mass of cells formed four to five days after fertilization is known as a blastocyst. The blastocyst contains a mass of inner cells known as the embryoblast and a mass of outer cells known as the trophoblast. The trophoblast becomes the placenta and surrounds a hollow cavity called the blastocoel and an inner cell mass (ICM), a group of cells at one end of the blastocoel that develops into the embryo proper. Pluripotent ES cells are derived from the ICM.
Stem cell differentiation.
Long before ES cells were cultivated in vitro, scientists were trying to understand regeneration. In the 1950s, Briggs and King developed a technique for replacing the nucleus of a frog's egg with the nucleus of another cell. In 1962, Gurdon used this technique to swap the nucleus of a fully differentiated small intestine cell into an egg to clone a frog. These experiments demonstrated that the changes seen in development are not due to permanent changes to the genetic code and that differentiation can occur in response to environmental factors. Subsequently, it was deduced that factors present in the oocyte must play a role in driving differentiation and development.
In the 1950s and 1960s, a tumor isolated from a mouse and known as a teratocarcinoma was studied in great detail. In 1964, Kleinsmith and Pierce demonstrated that these cells had unlimited self-renewal capabilities and multi-lineage differentiation, hence they were pluripotent. The establishment of stable cultures of these embryonic cancer (EC) cells by Kahan and Ephrussi (1970) was the precursor of ES cell culture. In the interim, EC cells were isolated from humans. Although these cells were useful for a variety of studies, most had limited therapeutic potential due to the genetic changes that occurred during tumor formation.
In the 1960s, McCulloch and Till conducted a series of experiments for measuring radiation sensitivity and used bone marrow cells for their transplantations. They observed spleen nodules containing dividing cells and subsequently traced their origin to a single stem cell (Becker et al. 1963, Siminovitch et al. 1963, Till et al. 1964). These early experiments form the basis for modern day adult and embryonic stem cell research.
Mouse ES (mES) cells were first cultivated successfully by Evans and Kaufmann (1981) and Martin (1981). The cells were derived from the ICM of mouse blastocysts and maintained in culture on a layer of mitotically inactivated mouse embryonic fibroblast cells known as "feeder cells" that provide optimal culture conditions.
The addition of other molecules known as cytokines (LIF and BMP-2) was found to enhance continued cell growth (see Yu and Thomson (2008) review).
Human ES (hES) cells were successfully cultivated and maintained in an undifferentiated state by Thomson et al. (1998). Borrowing from research done in primates (Thomson et al. 1995, 1996) as well as work done in culturing human in vitro fertilization (IVF) embryos (Gardner et al. 1998), Thomson's team developed conditions optimal for growth and maintenance of hES cells. While the feeder cell layer used in culturing mES cells was similarly employed for the cells, it was discovered that biochemical pathways and growth factors involved in hES maintenance were quite different from the mES cells. Recent advances in growth conditions have now eliminated the need for feeder cells in many cases, replacing them with an artificial matrix and more defined growth media. Cells grown under these conditions are karotypically normal, i.e. the chromosomes appear normal when stained, and even after prolonged proliferation in the undifferentiated state can give rise to each of the three cell types found in the embryo.
WiCEll (www.wicell.org) was formed in 1999 as a repository for human stem cell lines. As the National and International Stem Cell Bank, it grows, characterizes, and distributes hES cells and induced pluripotent stem (iPS) cells (see more below) to academic and industrial partners. The Stem Cell Unit at the National Institutes of Health also provides a database of federally approved stem cell lines against which any new hES or iPS cells can be compared. In Europe, the UK Stem Cell Bank (www.ukstemcellbank.org.uk) provides a repository of human stem cell lines. Its role is to provide stocks of these cells to researchers worldwide. The European Human Embryonic Stem Cell Registry (www.hescreg.eu), based in Berlin, provides the stem cell community with an in-depth overview on the current status of hES cell research in Europe. The Stem Cell Network – Asia-Pacific (SNAP) (www.asiapacificstemcells.org) was formed in 2007 with a mission of building a strong and dynamic stem cell research community in the Asia-Pacific region.
Stem cells can also be isolated from the adult organism. These cells, known as somatic stem cells, are multi or unipotent. They are found in different organs of the body, including the bone marrow, stomach, intestines, nose, and liver. Unlike embryonic stem cells, somatic stem cells differentiate into the cell type from which they originate. These cells are used by the body to maintain and repair the originating tissue. Scientists are still uncovering other areas of the body that contain these quiescent stem cells and are attempting to understand their role. Adult stem cells have been used extensively for research as well as clinical applications. For instance, bone marrow contains stem cells that are constantly replenishing the blood system with specific cells required for survival. Bone marrow transplants are a common treatment in patients with severe leukemia and other blood-related diseases. Recent efforts are focused on stem cells in the brain for treatment of Alzheimer's and related dementia disease (Taupin 2009) and in the heart for treatment of cardiac problems (Barile et al. 2009).
Hematopoietic cell differentiation.
All cells in the body, except for sex (germ) cells, contain approximately the same genetic material. In theory, if the "imprinting" that pushed a cell down a specific differentiation pathway could be erased, a cell could again become pluripotent. This imprinting can be caused by proteins such as transcription factors binding the DNA, the addition of molecules such as methyl groups to sites on the DNA, or the addition of methyl and acetyl groups or the molecule ubiquitin onto the histones. Removal of these changes can contribute to reverting the expression of genes in the cell so that it becomes pluripotent.
In 2006, Takahashi and Yamanaka demonstrated that by introducing four genes, Oct4, Sox2, Kfl4, and cMyc, into a differentiated mouse cell, the cell could become pluripotent. These "reprogrammed" cells were similar to mES cells, but they still contained some of the epigenetic patterning created through developmental differentiation. Additionally, these iPS cells did not support the development of viable chimeric mice upon injection into the blastocyst, the gold standard of the embryonic stem cell.
However, by the next year Yamanaka's group overcame this hurdle and began producing chimeric mice from their iPS cells (Okita et al. 2007). These exciting discoveries propelled a number of studies that sought to understand the factors that control cellular reprogramming, including how to identify cells that share similar properties with ES cells, which factors control epigenetic changes and which factors are absolutely necessary for reprogramming (Maherali et al. 2007, Okita et al. 2008, Mikkelsen et al. 2008). Human iPS cells were quick to follow (Wernig et al. 2007, Takahashi et al. 2007, Yu et al. 2007) as these details were revealed, and now human iPS cells have been created from such diverse sources as an 89-year-old patient with ALS (Lou Gehrig's disease) (Dimos et al. 2008) and a person suffering from Parkinson's disease (Soldner et al. 2009).
Originally iPS cells were generated using a viral construct to insert the genetic material into the chromosome. Although successful, the lack of control over the location of virally inserted material and the number of copies inserted makes viruses unsuitable for iPS cell production in the long run, so other methods were quickly developed. In 2009, Yu et al. eliminated the need for continuous presence of vectors and transgene sequences that were previously required for reprogramming by using nonintegrating episomal vectors to generate iPS cells. It has also been shown that direct DNA delivery of the genes or the proteins that are produced by these genes can induce a pluripotent state (Zhou et al. 2009). Studies involving generation of iPS cells using microRNAs and small molecules are underway (Huangfu et al. 2008, Judson et al. 2009). Other efforts (Nakagawa et al. 2008, Wernig et al. 2008) have shown that not all the genes described initially are required to induce pluripotency; in fact, at least one paper described that Oct4 alone was enough to reprogram neural stem cells (Kim et al. 2009). These discoveries are moving the use of iPS cells closer to clinical application.
Chimeric mice produced by injection of MEF iPS cells into heterologous blastocysts. Image courtesy of Dr Miguel Esteban, Stem Cell and Cancer Biology Group, Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510663, China.
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